DE102008009216A1 - Apparatus and method for spatially high resolution imaging of a structure of a sample - Google Patents

Abstract

Apparatus and method for spatially high-resolution imaging of a structure of a sample, in particular a microscope, characterized by a diffraction-limited resolution volume, with a plurality of switchable between different states dye molecules (UF), wherein at least one state is fluorescent, the fluorescence with a lens (O) collected and is imaged onto a spatially resolving detector with an optical system, wherein the UF in at least a portion of the sample has a distribution density greater than the inverse of the diffraction limited resolution volume, one or more light sources to emit a switching radiation to form a first subset of the UF in to switch the sample, and to emit an excitation radiation to excite the first subset of the UF, wherein at least one of the light sources is arranged so as to irradiate the sample and a switching and / or fluorescence excitation of the UF in the sample at least in one direction approximately perpendicular to the optical axis and in particular in the focus of the objective (O), wherein advantageously the switching is a photoactivation or deactivation of the UF and the light source for switching and / or the light source for excitation a focusing device for generating a in Direction of the illumination extended, at least in one direction at least approximately perpendicular to the optical axis of the lens line-like ...

Description

State of the art

• [1] Stelzer et al., Optics Letters 31, 1477 (2006).
• [2] Stelzer et al., Science 305, 1007 (2004).
• [3] DE 102 57 423 A1
• [4] WO 2004/0530558 A1

The so-called SPIM technology (Selective Plane Illumination Microscopy) is described, for example, in Stelzer et al. [1-4] has been described as a microscopy method:

• [1] Stelzer et al., Optics Letters 31, 1477 (2006).
• [2] Stelzer et al., Science 305, 1007 (2004).
• [3] DE 102 57 423 A1
• [4] WO 2004/0530558 A1

As confocal laser scanning microscopy allows SPIM as a widefield technique the inclusion of 3D objects in the form of optical sections, the Advantages above all in the speed, the small fading the sample as well as an extended penetration depth are to be sought. As a rule, fluorophores in the sample are irradiated with laser light stimulated in the form of a light sheet. The light sheet can through the Sample will be scanned.

By the (computational) combination of images that come from different Angles are recorded, can be a spherical Create PSF. Their extension is usually through the lateral Resolution of the detection objective used determines which overall the achievable optical resolution in the limited to conventional SPIM method.

In Breuninger et al., Optics Letters, Vol. 13, 2007 the SPIM method is described with a periodically structured light sheet. The periodically structured light sheet fluorescence excitation takes place at the high intensity locations. The patterning is used to suppress scattered light from out-of-focus planes and to increase resolution through structured illumination (see below in the text). Heintzmann et al. ).

In WO2006 / 127692 is the so-called PALM method (photoactivated light microscopy) described. The method is based on the photoactivation of individual molecules separated according to the extents of the detection PSF and their highly accurate localization by fluorescence detection.

• 1.) Photoaktivierung von Einzelmolekülen: Durch die Aktivierung werden die Fluoreszenzeigenschaften der Moleküle geändert (Ein-/Ausschalten, Änderung des Emissionsspektrum, ...), wobei die Aktivierung so erfolgt, dass der Abstand zwischen aktivierten Molekülen größer gleich der optischen Auflösung des Standardmikroskops (gegeben durch Abbesches Auflösungsgrenze) ist.
• 2.) Anregung der aktivierten Moleküle und Lokalisieren der Moleküle mit einem ortsauflösenden Detektor.
• 3.) Deaktivieren der aktivierten Moleküle.
• 4.) Wiederholung der Schritte 1–3 und Überlagerung der Lokalisationspunkte aus Schritt 2.), die aus verschiedenen Iterationschritten gewonnen wurden zu einem hochaufgelösten Bild.

The PALM method as in WO2006 / 127692 essentially uses the following primary steps to produce a microscopic image with an increased optical resolution compared to the standard microscope:

• 1.) Photoactivation of single molecules: Activation changes the fluorescence properties of the molecules (switching on / off, changing the emission spectrum, ...), whereby the activation takes place so that the distance between activated molecules is greater than or equal to the optical resolution of the standard microscope (given by Abbe's resolution limit).
• 2.) excitation of the activated molecules and localization of the molecules with a spatially resolving detector.
• 3.) deactivating the activated molecules.
• 4.) Repeat steps 1-3 and overlay the localization points from step 2.), obtained from different iterations to a high-resolution image.

The activation preferably takes place in wide-field illumination and distributed statistically. The choice of the activation energy is an attempt to achieve that as few / no molecules (1) with overlapping diffraction slices (2) on the camera arise (see 1a ). However, overlapping diffraction disks are accepted and can not be evaluated ((3) in 1b ). This results in regions in which the distance between the activated molecules is larger or much larger than the diffraction disc on the camera (4). Due to the statistical activation of the molecules, about 10,000 individual images must be evaluated for the generation of a high-resolution image to determine the positions of the molecules. As a result, large amounts of data must be processed and the measurement slowed down (about 1 min per high-resolution image). The billing of the individual images to a high-resolution image requires about 4 hours.

The Application of the PALM method in 3D imaging presents difficulties as well as activating molecules outside the focal plane and be bleached without their fluorescent light in the Imaging could be exploited. Above all, will be at biological samples throughout the focus cone autofluorescent light stimulated, which is to be regarded as an interference signal and the Extremely low contrast.

hereby The recording of a z-scan is prevented, so no 3D image the sample can be obtained.

To avoid the photoactivation and disturbing autofluorescence outside the focal plane is in the WO2006 / 127692 described the use of multiphoton excitation. However, this arrangement is technically complicated. For example, the dyes (PA-GFP) must be non-linearly activatable and high intensities must be used which can lead to dye damage or sample damage.

Another method used to avoid the autofluorescence problem is the Kom Combination of the PALM method with the TIRF technology, where the excitation volume in the z-direction is kept very small due to the restriction to evanescent waves. With TIRF, however, no 3D imaging is feasible.

Basically, PALM offers only an improvement of the lateral resolution due to the spatially resolved detection. The axial resolution is determined primarily by the extent of the detection PSF used. This is another reason for the combination of PALM with the TIRF technology, which offers a high axial resolution (see also WO2006 / 127692 ).

Next PALM, other resolution-enhancing methods are known, in which the sample is illuminated so that one by fluorescence Detectable region arises, which is smaller than the diffraction limit according to Abbe.

• • Abregung zuvor angeregte Moleküle durch stimulierte Emission ( STED, Klar and Hell, Opt. Lett. 24 (1999) 954–956 )
• • Abregung zuvor angeregte Moleküle durch Weiteranregung in einen höheren nicht fluoreszenzfähigen Zustand ( Excited State Absorption, Watanabe et al., Optics Express 11 (2003) 3271 )
• • Entvölkerung des Grundzustandes durch Tripletbesetzung ( Ground State Depletion, Hell and Kroug, Appl. Phys. B 60 (1995), 495–497 )
• • Schalten eines Farbstoffs zwischen einem fluoreszierenden und nicht fluoreszierenden, weniger fluoreszierenden oder anders (wie andere Emissionswellenlänge, Polarisation) gekennzeichneten fluoreszierenden Zustand ( Hell, Jakobs, and Kastrup, Appl. Phys. A 77 (2003) 859–860 )

This is achieved by a nonlinear interaction according to different methods:

• Depletion of previously stimulated molecules by stimulated emission ( STED, clear and bright, Opt. Lett. 24 (1999) 954-956 )
• Depletion of previously excited molecules by further excitation to a higher non-fluorescent state ( Excited State Absorption, Watanabe et al., Optics Express 11 (2003) 3271 )
• • depopulation of the ground state by triplet occupation ( Ground State Depletion, Hell and Kroug, Appl. Phys. B 60 (1995), 495-497 )
• Switching a dye between a fluorescent and non-fluorescent, less fluorescent or otherwise (like other emission wavelength, polarization) marked fluorescent state ( Hell, Jakobs, and Kastrup, Appl. Phys. A 77 (2003) 859-860 )

In usually these are point-scanning methods that Disadvantages in terms of a fast data recording offer. moreover the sample becomes unnecessary in extra-focal areas loaded.

Heintzmann et al. ( R. Heintzmann, TM Jovin and C. Cremer, "Saturated patterned excitation microscopy - a concept for optical resolution improvement", JOSA A 19, 1599-1609 (2002) .) propose a nonlinear process in the form of direct saturation of a fluorescence transition as another concept for increasing the resolution. The increased resolution is based on a periodically grid-like patterned illumination of the sample, whereby a transfer of high spatial frequencies of the object takes place in the range of the optical transfer function of the microscope. The transfer can be followed indirectly by theoretical post-processing of the data. It is also disadvantageous in these methods that the sample is unnecessarily stressed in non-focal areas, since the structured illumination penetrates the entire sample space. In addition, the method is currently not applicable to thick samples, since the fluorescence excited out of focus as a background signal on the detector and thus reduces the dynamic range.

Object and presentation the invention

task The invention is the disadvantages of the methods described above to avoid. The invention describes methods and arrangement for Realization of a PALM microscope with optimized photoactivation to realize a higher frame rate. Across from PALM will be a high resolution 3D imaging with no nonlinear Photoactivation achieved. A PALM TIRF combination for reduction the extra-focal autofluorescence is not required.

It can the beneficial use of the MultiView method (several Illumination angle on the sample) to achieve an increased Penetration depth and an isotropic optical release in x, y, z take place.

There is an activation accordingly 2 as many molecules as possible without "gaps" ((4) in 1b ) and without the diffraction disks of the molecules being superimposed on the camera ((3) in 1b ). Substituting the diffraction discs of the individual molecules to each other so preferably results in a dense sphere packing.

hereby will increase the speed of the PALM process and achieved a reduction in the number of frames.

The arrangements according to the invention and their effects and advantages will be apparent from the 3 - 7 described in more detail. According to the invention, a surprisingly advantageous combination of the SPIM method with the PALM method is advantageously proposed.

3 schematically shows from the side (direction of incidence equal viewing direction) a sheet-like light sheet LB, also called a lens, which is generated for example with a cylindrical lens (O2 - ZL) and passes through the sample (P).

Above The sample is an objective O of a microscope, for example a wide-field microscope with a CCD camera, a laser scanning microscope or a microscope with structured illumination.

By the side of the sample irradiated light sheet LB in the form of the SPIM light sheet, the Substantially located exactly in the focal plane (F) of the lens O takes place in 3 a photoactivation perpendicular to the optical axis of fluorescence excitation and detection. The fluorescence excitation can take place here only within the focal plane (F). Fluorescence excitation and fluorescence detection are performed according to WO2006 / 127692 via the microscope objective O. Thus, a localized excitation in the z-direction takes place and samples can be examined three-dimensionally without nonlinear photoactivation with the PALM method.

The Width of the light beam for photoactivation, d. H. its extension in z-direction, is adapted so that it is advantageously smaller or equal to that given by the numerical aperture of the objective O. axial extent of the PSF. This advantageously prevents that fluorescence molecules outside the focal plane be activated and blanched. In addition, a fluorescence only from this plane defined by the activation beam come. Thus, this arrangement is inherently 3D-resolving.

The Detection takes place by conventional means, such as by classical wide field microscopy, confocal microscopy or structured lighting (ZEISS APOTOM).

However, there is the problem here that the excitation beam is able to excite autofluorescence over the entire sample space. This can be prevented in 3 In addition, the light beam for excitation of the fluorescence (after photoactivation) is irradiated with a light sheet (LB) perpendicular to the detection via O2 - ZL. This ensures that no out-of-focus autofluorescence signals are generated that interfere with imaging. In addition, the fluorescent light can be detected particularly efficiently separated from the excitation light, since no spectral separation by means of a dichroic beam splitter is required. For some switchable dyes, such as DENTRA, photoactivation and fluorescence excitation occur at the same wavelength. This can be realized particularly easily hereby.

It Furthermore, an arrangement is conceivable in which the photoactivation via the lens O. and the fluorescence excitation via the side irradiated light sheet O2 takes place. The lens O is still used for Detection. Here, too, becomes extra-focal autofluorescent light avoided. Extra-focal autofluorescence, which is caused by the Activation beam is generated, can be spectrally, but especially in time (the fluorescence excitation occurs after activation) of the fluorescence signal of interest separate.

One Problem with this method is the generation of non-focal It is therefore in this method advantageous to use molecules that are absorbed after recording an image plane over the entire sample area z. B. by a Deactivate far-light illumination.

The recording of the high-resolution image is carried out for the inventive arrangements as in WO2006 / 127692 described with the above-mentioned steps 1-4. The activatable fluorescent dyes used are preferably fluorescent proteins known from the prior art, such as PA-GFP or DRONPA. The photoactivation takes place here with 405 nm, the fluorescence excitation at 488 nm and the detection in the range above 490 nm. Furthermore, reversibly switchable synthetic dyes such as Alexa / Cyan constructs can be used.

In 4a schematically another arrangement according to the invention with a lighting by means of two light sheets LB 1 and LB 2 is shown. The interferometric superimposition of light sheets from several directions (but in the focal plane defined by the detection) results in an interference pattern along the marked x-direction in the focal plane. The light sheets may in turn contain laser light for photoactivation and / or fluorescence excitation. Due to the superimposition, a so-called standing wave field (SW) forms, which is schematically shown in FIG 4b is shown as a striped pattern. If, for example, two sheets of light are used for the photoactivation, it can be achieved that the spacing of the fluorescence emitters to be activated is greater than or equal to the width of the PSF of the detection (O in FIG 4a ). As a result, preferably no fluorescence emitters spatially superimposed along the x-direction, which are additionally located in the z-direction at the location of the focal plane F. The detection is again analogous to the arrangement according to 3 , A displacement of the standing wave field in the focal plane to illuminate the sample in the intensity minima is done by adjusting the relative phase between the two sheets, for example with a phase modulator (PH).

When 2 beams are used, interference causes stripe-shaped activation located in x and z. Upon irradiation of more than two, advantageously three sheets LB 1 to LB 3 (120 ° angle) is formed as in 5 12 illustrates a dot pattern PM, that is, an activation located in x, z, and y such that the distance (1) of the activated fluorescence emitters is greater than or equal to the width of the PSF of the detection (O in FIG 4a ). The fluorescence excitation can preferably also take place via one or more light sheets, which in turn avoids extra-focal autofluorescence.

Fluorescence excitation from direction O in 4a can also be structured in one or more directions and, for example, also have a hole pattern (structuring in x and y). In this way it can be ensured that the distance of the fluorescence emitters is greater than or equal to the width of the PSF of the detection. Such structuring can be carried out, for example, with a lattice structure ( DE 10257237A1 ) or with a multispot excitation ( DE 10 2006 017 841 ) respectively.

Farther photoactivation can be done via the lens O. and the fluorescence excitation can be realized with a plurality of light disk beams, which form an interference pattern of the type described above. Molecules with overlapping diffraction slices activated, are stimulated to different degrees. Also In this way, gaps in the camera image are avoided. By the activation beam generated autofluorescence leaves Separate temporally and / or spectrally. After taking a Layer needs to be disabled for 3D shooting take place the sample area.

If the photoactivation is carried out by the objective O, the structured activation can also be realized by a specific imaging (eg of a grating - as described above) or by a scanning mechanism. For this purpose, the light beam can, for example, scan the image field and its intensity during the movement is changed by, for example, a fast AOTF such that an activation pattern corresponds, for example, accordingly 5 arises in the focal plane. In this method, however, molecules are also activated outside the focal plane. By a lateral light sheet fluorescence excitation can be ensured that still only fluorescence is detected from the focal plane. However, once a plane has been captured, it must be disabled for 3D imaging over the sample area. The intensity of the activation beam is ideally chosen so that on average only one molecule per activation spot (equivalent to approx. PSF size) is excited. This reduces the likelihood that 2 molecules with overlapping diffraction slices will be activated simultaneously. In the course of image acquisition, the activation pattern must of course be phase-shifted, so that all molecules are activated evenly. Autofluorescence generated by the activation beam can be separated temporally and / or spectrally. The activation intensities are usually so small that autofluorescence should not play a role.

Of the Photoactivation and fluorescence excitation beam can be exchanged here. This has the advantage of being outside the focal plane no photoactivation happens. This can be the activation unstructured over the light sheet and the excitation on the PSF matched structured over the lens performed become. Molecules with overlapping diffraction slices activated, are stimulated to different degrees. On too this way, gaps in the camera image can be avoided become. However, there is the problem of the extra-foci Autofluorescence.

One Problem with all variants described is the axial resolution in the SPIM process generally by the width of the light sheet used is determined. As the NA used to generate it usually much smaller than the NA of the detection lens is directly address the problem of a highly elongated system PSF (lateral Extension determined by the resolution of the PALM method (Nanometer range), axial extent determined by the leaf width (Micrometer range). This causes disadvantages in 3D imaging. With the help of known from the prior art multiview technology (Picking up stacks from different angles) may cause this problem be bypassed and an effective, largely homogeneous spatial Resolution according to the lateral PALM resolution be generated.

It proves to be particularly advantageous if the photoactivated molecules in the edge regions of the light sheet used for activation are deactivated again by a further structured light sheet in the sense of a nonlinear interaction in order to achieve a higher z resolution. This can be done by one of the methods described above, preferably a switching process. A light sheet may also be used as the deactivation beam, but is structured in such a way that it has a zero point in the focal plane over the area of the image area to be observed, as in FIG 6 shown. The pupil intensity distribution ( I ) of the light-beam corresponds here to a line, which was previously generated by a suitable optics (eg with Powell lens). In the pupil is a phase plate ( II ), which covers one half of the line ( III ) which produces a pi-phase jump. The light sheet extended in the xy plane ( V ) is replaced by a suitable optic ( IV ) generated. This results in the range of the depth of field of the illumination optics ( IV ) a zero level ( VI ) parallel to the focal plane of the detection optics ( VII ), in which no molecules are de-energized. According to the STED-like methods, this can severely restrict the axial extent of the light sheet with photoactivated molecules in a nonlinear manner and improve the axial resolution.

7 shows a general optical out Example of the use of the described advantageous methods and applications with photoactivation / deactivation on (x-direction structured) light sheet and CCD wide-field detection.

The sample is marked, for example, with Dronpa and can be turned on (activated) above 405 nm and excited at 488 nm or switched off again. The lasers ( 1 ) are provided for 405 nm (photoactivation) and 488 nm (fluorescence excitation and photodeactivation) and are transmitted via a beam union ( 2 ) and dichroic mirrors united. For photoactivation and fluorescence excitation, it is also possible to use one and the same wavelength as explained above using the example of DENTRA. This allows a laser (405 nm in this case) and the beam combination ( 2 ) accounted for.

An AOTF ( 3 ) is used for wavelength selection and fast switching / attenuation of the laser wavelengths. It is preferably a rotatable lambda / 2 plate in the illumination direction ( 4 ) and a pole splitter (PBS) ( 5 ) arranged downstream with fiber coupling for 2 channels. By turning the lambda / 2 plate, the power in the two channels can be adjusted.

About the single-mode fibers ( 6 ) the light is transmitted via cylindrical optics ( 7 ) for generating a light sheet and imaging optics ( 8th ) via optical paths ( 10 ) respectively. ( 11 ) for photoactivation (or deactivation) on the sample ( 12 ). For generating zeros in the focal plane (x-patterning) according to 4 the optical path lengths are adjusted accordingly. A displacement of the standing wave field in the focal plane to illuminate the sample in the intensity minima is done by adjusting the relative phase between the two sheets, for example with a phase modulator (PH).

That in the sample ( 12 ) is transmitted via a detection objective ( 13 ) (Microscope objective) in a detection beam path ( 14 ) via a tube lens ( 15 ) and emission filters ( 16 ) detected by means of a CCD camera. Dashed in the detection beam path is an optional (swivelable) color splitter ( 18 ) for the reflection of a laser ( 22 ) or a wide field light source ( 23 ), if the fluorescence excitation is effected by the detection objective. Between laser and wide-field light source can optionally be adjusted with the help of a retractable mirror ( 20 ) to get voted. The laser ( 22 ) or the wide field light source ( 23 ) can also be used for activation, in which case instead of a wavelength of 488 nm, a wavelength of 405 nm is to be provided when DRONPA is used as the dye. In particular, for the laser beam path ( 22 ) a scanner unit ( 21 ), which allows a punctiform scanning of the image field. Between the scanner unit ( 21 ) and the color divider ( 18 ) is an adapted imaging optics ( 19 ) intended.

For the above based on 6 described z-structuring of the light sheet can phase plates ( 9 ) are introduced into the illumination beam paths.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - DE 10257423 A1 [0001]
• WO 2004/0530558 A1 [0001]
• - WO 2006/127692 [0005, 0006, 0010, 0012, 0024, 0030]
• - DE 10257237 A1 [0033]
• - DE 102006017841 [0033]

Cited non-patent literature

• Stelzer et al., Optics Letters 31, 1477 (2006). [0001]
• Stelzer et al., Science 305, 1007 (2004). [0001]
• Breuninger et al., Optics Letters, Vol. 13, 2007 [0004]
• Heintzmann et al. [0004]
• - STED, clear and bright, Opt. Lett. 24 (1999) 954-956 [0014]
• Excited State Absorption, Watanabe et al., Optics Express 11 (2003) 3271 [0014]
• Ground State Depletion, Hell and Kroug, Appl. Phys. B 60 (1995), 495-497 [0014]
• - Hell, Jakobs, and Kastrup, Appl. Phys. A 77 (2003) 859-860 [0014]
• Heintzmann et al. [0016]
• R. Heintzmann, TM Jovin and C. Cremer, "Saturated patterned excitation microscopy - a concept for optical resolution improvement", JOSA A 19, 1599-1609 (2002) [0016]

Claims (38)

Device, in particular a microscope, characterized by a diffraction-limited resolution volume, With several switchable between different states Dye molecules (UF), wherein at least one state fluorescent is, the fluorescence collected with a lens (O) and with a optical system imaged on a spatially resolving detector becomes, wherein the UF in at least a portion of the sample a Have distribution density that is greater than that Inverse of the diffraction-limited resolution volume; one or several light sources for emitting a switching radiation, to toggle a first subset of the UF in the sample and to Emitting an excitation radiation to the first subset of To stimulate UF, in which at least one of the light sources in such a way is arranged to irradiate the sample and a switch and / or fluorescence excitation of the UF in the sample at least in one Direction approximately perpendicular to the optical axis and in particular in the focus of the objective (O). Apparatus according to claim 1, wherein the switching a photoactivation or deactivation of the UF. Device according to at least one of the preceding Claims, wherein a driving unit for control the switching radiation is provided to ensure that the distribution density is in the fluorescent state UF is less than the inverse of the diffraction-limited resolution volume the device is. Device according to at least one of the preceding Claims, wherein for the light source for switching and / or the light source for exciting a focusing arrangement for Generation of one in the direction of lighting extended at least in one direction at least approximately perpendicular to the optical Axis of the lens line-like illumination area provided is. Device according to at least one of the preceding Claims, wherein a flat sheet of light with the focusing arrangement is generated, which irradiates the sample and at least approximately perpendicular to the optical axis of Lens is. Apparatus according to claim 5, wherein the focusing arrangement is an astigmatic and / or aspheric optic. Device according to at least one of the preceding Claims, wherein the used for switching and stimulation Light sources in a wavelength switchable Light source are united. Device according to at least one of the preceding Claims, wherein the used for switching and stimulation Light sources have the same wavelength. Device according to at least one of the preceding Claims, wherein different light sources with one wavelength are provided for switching and stimulation. Device according to at least one of the preceding Claims, wherein a plurality of light sources for excitation and / or Switching from different directions are provided. Device according to at least one of the preceding Claims wherein two opposing ones Light sources are provided. Device according to at least one of the preceding Claims, with at least three from different directions Incident light sources are provided. Device according to at least one of the preceding Claims, wherein multiple light sources by splitting the light of a light source are formed. Device according to at least one of the preceding Claims, wherein a plurality of light sources for generating Interference have the same wavelengths. Device according to at least one of the preceding Claims, wherein at least one light source radiated laterally and suitable optical means are provided, which leads to a Structuring lead in the direction of detection. A method for spatially high-resolution imaging of a structure of a sample, comprising the steps of: selecting a substance from a group of substances which can be converted with a switching signal at least once from a first state with first optical properties into a second state with second optical properties. Transferring changing proportions of the substance with the switching signal in the second state, wherein the switching takes place so that the distance between switched molecules is greater than or equal to the diffraction-limited optical resolution of the microscope. Stimulating the transferred to the second state An parts and localization of the molecules with a spatially resolving detector spatially resolved registering an optical measurement signal via a lens with a detector emanating from the dye molecules located in the second state, wherein at least the transition to the second state and / or the excitation at least approximately takes place perpendicular to the detection direction and / or to the optical axis of the lens. The method of claim 16, wherein a deactivation of the molecules from the second state to the first state he follows. Method according to at least one of the preceding Claims, wherein for the light source for transfer in the second state and / or the light source for excitation by Focusing one towards the lighting at least in one Direction linearly extended illumination area takes place. Method according to at least one of the preceding Claims, wherein the illumination area is a linear Represents light distribution in the field of view of the lens. Method according to at least one of the preceding Claims, wherein the illumination area is a planar Light leaf in the field of view of the lens represents. Method according to at least one of the preceding Claims, wherein a transfer in the second state by photoactivation. Method according to at least one of the preceding Claims wherein excitation by fluorescence excitation he follows. Method according to at least one of the preceding Claims, wherein at least the photoactivation and / or the fluorescence excitation perpendicular to fluorescence detection done. Method according to at least one of the preceding Claims, in which 1. A photoactivation of Single molecules take place by the fluorescence properties the molecules are changed, with activation so that takes the distance between activated molecules greater than the optical resolution the microscope is, 2. An excitation of the activated molecules and localizing the molecules with a spatially resolving one Detector takes place, 3. Deactivation of the activated molecules he follows and 4. a repetition of steps 1-3 takes place, whereby by superposition of detection images a high-resolution image is composed. Method according to at least one of the preceding Claims, in which 1. A photoactivation of Single molecules via at least one light sheet takes place by the fluorescence properties of the molecules to be changed 2. Deactivation of molecules by means of a light sheet structured in the z-direction, so that the layer of activated molecules in z-direction less extensive than Abbe's resolution capacity the detection optics or the numerical aperture of the illumination optics corresponds, 3. An excitation of the activated molecules and localizing the molecules with a spatially resolving one 4. Detection of the activated molecules he follows and 5. a repetition of steps 1-4 takes place, whereby by superposition of detection images a high-resolution image is composed. Method according to at least one of the preceding Claims, wherein the activation and / or stimulation of a Sample area from one side. Method according to at least one of the preceding Claims, wherein the activation and / or stimulation of a Sample area is made from opposite sides. Method according to at least one of the preceding Claims, wherein the activation and / or stimulation of a Sample area of more than two sides, preferably of 3 sides he follows. Method according to at least one of the preceding Claims, wherein a plurality of light sources for generating Interference have the same wavelengths. Method according to at least one of the preceding Claims, wherein by two light sources and interference a stripe pattern is generated in the sample. Method according to at least one of the preceding Claims, wherein at least three light sources and Interference a dot pattern is generated in the sample. method according to at least one of the preceding claims. Method according to at least one of the preceding Claims, wherein multiple light sources by splitting the light of a light source are formed. Method according to at least one of the preceding Claims, wherein a stimulation by the lens and the Activation takes place via the light sheet. Method according to at least one of the preceding Claims, with stimulation and activation over the light sheet takes place. Method according to at least one of the preceding Claims, wherein the excitation light structuring having. The method of claim 36, wherein structuring is produced by a grid arranged in the excitation beam path. Method according to at least one of the preceding Claims, wherein the excitation light is a point distribution (Multispot). Method according to at least one of the preceding Claims, wherein the activation by the lens and the excitation is via the light sheet and the activation light by a modulated scanning movement or a grating image a point distribution having.